Exploring the influence of microgravity on chemotherapeutic drug response in cancer: Unveiling new perspectives

Abstract Microgravity, an altered gravity condition prevailing in space, has been reported to have a profound impact on human health. Researchers are very keen to comprehensively investigate the impact of microgravity and its intricate involvement in inducing physiological changes. Evidenced transformations were observed in the internal architecture including cytoskeletal organization and cell membrane morphology. These alterations can significantly influence cellular function, signalling pathways and overall cellular behaviour. Further, microgravity has been reported to alter in the expression profile of genes and metabolic pathways related to cellular processes, signalling cascades and structural proteins in cancer cells contributing to the overall changes in the cellular architecture. To investigate the effect of microgravity on cellular and molecular levels numerous ground‐based simulation systems employing both in vitro and in vivo models are used. Recently, researchers have explored the possibility of leveraging microgravity to potentially modulate cancer cells against chemotherapy. These findings hold promise for both understanding fundamental processes and could potentially lead to the development of more effective, personalized and innovative approaches in therapeutic advancements against cancer.

5][6] Gaining a comprehensive understanding of the cellular response under microgravity holds immense potential for various applications, including cancer biology and potentially leading to the discovery of novel approaches for cancer treatment.
Cancer, a highly intricate and diverse disease condition characterized by the uncontrolled proliferation and division of abnormal cells. 7It exhibits distinct characteristics such as autonomous growth, resistance to the signals that govern cell division, unrestrained replication, resistance to apoptosis, continuous angiogenesis and ability to invade and metastasize to distant tissues. 8cent studies have investigated the effect of microgravity conditions on the growth and progression of cancer cells. 2 in vitro studies utilizing ground-based simulation systems have highlighted the impact of microgravity on cancer cells, demonstrating altered metastatic potential in various cancer types including lung cancer and melanoma cells.Lung cancer cells exposed to simulated microgravity (SMG) exhibited decreased gene expression, resulting in a decrease in their ability to metastasize. 9,10emotherapy is a common modality used to target and treat cancer; however, cancer metastasis is the key cause of cancer therapy failure, leading to death in over 90% of cancer cases. 11tastasis, a multifaceted process, encompasses the dissemination of cancer cells from their primary location to distant regions of the body.It occurs across different cancer types, despite the vast diversity in their molecular biology, development and prognosis. 12is review delves into the impact of microgravity on the efficacy of chemotherapy and its effect on cancer cells.By exploring the microgravity influence on the efficacy of anticancer treatment, this report offers insights crucial for optimizing the use of chemotherapeutics agents and developing more effective strategies for combating cancer.

| APPROACHE S TO S IMUL ATING M I CRO G R AV IT Y
To study the impact of microgravity on biological systems, researchers have employed ground-based simulation systems that replicate space-like microgravity conditions.These simulation systems provide valuable insights into gravity-dependent phenomena using a wide range of organisms.However, each simulator has its own limitations and potential artefacts, which can inadvertently introduce unintended effects and distort the desired microgravity response. 13nsequently, the misinterpretation of responses to these side effects as specific microgravity effects can occur.Furthermore, when comparing results from experiments conducted on different simulators, inconsistencies or even conflicting responses may arise.
Hence, it is crucial to critically evaluate the physical parameters and principles of each simulator as well as their specific impact on biological processes and organisms of varying sizes.Not all simulators and operational modes are equally suitable for accurately simulating microgravity across all processes and organisms.Simulation of microgravity can be performed for both in vitro and in vivo studies to understand microgravity-associated alterations at the cellular level.

| Free fall machine (FFM)
In FFM (Figure 1), the sample is passed through an elongated tube, which ensures free fall of the sample for a few milliseconds before being acted upon by an air current that launches the sample upwards.The FFM creates a state of weightlessness as the sample attains terminal velocity during free fall, causing the sample (cells) to not experience gravitational force, resulting in weightlessness. 14,15

| Clinostats
Clinostats, also referred to as random positioning machine (RPM), is a commonly utilized equipment to simulate microgravity. 16inostats rely on the principle of clinorotation wherein centrifugal forces are generated whose intensities depend upon the distance of the sample (cells) from the rotation axes of the clinostat. 17rthermore, based on the number of rotation axes, clinostats can be categorized into 1D/2D and 3D (three-dimensional) systems.In 1D system (Figure 2A), the sample is rotated in one axis (vertical axis).In this position, the object experiences a gravity pull towards its lower side. 18In a 2D clinostat (Figure 2B), the sample (cells) is rotated about the horizontal axis via a rotary device in a continuous fashion, causing the gravity vector to change with respect to the constantly changing direction of the sample in rotation. 19By selecting an optimal rotational speed (10-20 rpm) with minimal centrifugal force, the sample experiences a microgravity environment. 20In the 3D clinostat (Figure 2C), the frames operate at constant speed and direction and consist of two perpendicular frames (gimbal-mount), one inside the other.These frames can rotate independently. 21The orientation of the gravity vector is continuously altered, resulting in an averaged gravity vector that mimics a microgravity environment. 221.3| Rotary cell culture system (RCCS) RCCS (Figure 3) is a novel technique designed for the cultivation of anchorage-dependent or suspension cells.The system consists of horizontally rotated culture vessels, which provide an environment for cell growth.During the culture process, the rotation speed can be customized to counteract cell sedimentation.The RCCS provides a unique setting with low shear forces, efficient mass transfer and SMG conditions.23

| Diamagnetic levitation
Diamagnetic levitation (Figure 4) employs a magnetic adjustable gravity simulator, which employs an intense magnetic field to generate forces that oppose the gravity force.Diamagnetic materials such as water and organic materials are repelled upwards or downwards depending on their position in the magnet.By regulating the magnetic field in the system microgravity conditions can be simulated.
Levitation is achieved when the magnetic forces balance the weight of the material and create a weightless environment by reducing internal gravity-induced stress. 24This technique can simulate partial gravity by placing samples at different points in the magnetic field gradient and produce intermediate gravitational forces similar to those of the space environment. 25Diamagnetic levitation is extensively used to investigate the effect of microgravity in vitro experiments.

| Simulated microgravity for in vivo systems
To investigate the impact of microgravity on physiological processes, cellular and biochemical parameters in in vivo systems, researchers employ ground-based simulation techniques.Currently, researchers employ hindlimb unloading and tail suspension models (Figure 5A and Figure 5B) to study the impact of microgravity on mice and rat models.In these models, the body of the animal makes an approximately 30° angle from the floor of the cage such that the animal does not touch the grid floor with its back feet. 26,27is suspension ensures the removal of mechanical loading from the hindlimbs simulating microgravity conditions. 28

| Head-down tilt method
In the HDT method (Figure 6) the subjects are positioned in a recline position on an inclined bed tilted at a 6° angle with the head closer to the ground and feet elevated.HDT induces a headward fluid shift of blood from the lower body to the upper body causing hydrostatic pressure in blood vessels. 29HDT methods are best employed in studying various physiological systems, musculoskeletal and cardiovascular disorders. 30HDT methods can provide valuable insights into understanding the effect of SMG.

| Dry immersion method
The dry immersion method is another widely used ground-based model for studying the effect of microgravity (Figure 6).In the dry immersion method, the subjects are submerged in water up to the neck, inducing a floating sensation that stimulates the microgravity effect. 31Dry immersion also induces a headward fluid shift, similar F I G U R E 1 Schematic representation of a free fall machine (FFM).
to the condition experienced in space.Dry immersion offers a valuable method for studying the physiological responses to microgravity, however, its limitations should be considered when interpreting the results. 29,31

| Wet Immersion method
The water immersion method involves submersion into water from the neck down (Figure 6). 32In the wet immersion method, the subjects are immersed in water, which exerts hydrostatic pressure causing redistribution of body fluids towards the upper body, simulating the fluid shifts that occur in microgravity. 33

| Unilateral lower limb suspension (ULLS) method
ULLS is a cost-effective method used to study the effect of microgravity on the human body (Figure 6).It involves elevating one leg using a tall boot and crutches such that one foot is not in contact with the ground and allowing the subject to maintain mobility. 27In the ULLS method, the hydrostatic pressure is absent.This method provides valuable insights into localized musculoskeletal adaptations similar to those observed during space travel. 34However, ULLS is not ideal for evaluating the global effect and long-term impact of microgravity. 29

| Supine bed rest method
In the supine bed rest method, the subjects lie in a supine position for an extended period of time (Figure 6).This method simulates some of the physiological changes that occur during spaceflight or extended duration of inactivity, such as reduced physical activity and fluid distribution. 30The supine bed rest model allows the researcher to investigate the effect of prolonged immobility on various physiological systems including alterations in metabolic processes and musculoskeletal changes. 35By comparing the results of subjects exposed to supine bed rest to those in the space environment, researchers can gain insight into the specific effect of microgravity on the body. 29

| C AN CER PROG RE SS I ON
Cancer metastasis can occur via various modes that involve epithelial-mesenchymal transition (EMT), 36 accumulation of mutations in stem cells 37 and tumour-associated macrophages promoting metastasis progression. 38Genetic mutations play a critical role in the development and progression of metastatic cancer.Studies have reported that mutations in genes such as PTEN (Phosphatase and TENsin homologue deleted on chromosome 10), CDKN2A (cyclin-dependent kinase inhibitor 2A), TP53, KRAS (K-Ras), SMAD4 (Mothers against decapentaplegic homologue 4) and BRCA1/2 (Breast cancer 1 and 2) have been associated with metastatic cancer.The development of cancer involves four stages initiation, promotion, progression and metastasis, each marked by distinct molecular and cellular alterations (Figure 7).The initiation phase is a critical phase that involves genetic alterations resulting in the imbalance of biochemical signalling pathways that regulate cellular processes leading to pre-cancerous cells. 40In the promotion phase, the pre-cancerous cells are stimulated by promoting factors to create a favourable environment to develop into preneoplastic lesions or benign tumours leading to malignant tumours. 41Malignant tumours evade normal cellular control and instigate invasion through the degradation of extracellular matrix (ECM) by matrix metalloproteins (MMPs).The process of tumour invasion is further driven by cytokines such as interleukins (IL-6 and IL-8), genetic alteration in the tumour suppression genes (ras family and p53 tumour suppressors genes) and compromised DNA repair machinery. 8,42,43Another critical factor is angiogenesis, driven by hypoxia-inducible factor (HIF) which stimulates vascular endothelial growth factor (VEGF) and promotes cancer progression. 44e final stage of cancer development is metastasis, where the cells disseminate from the site of origin to distant organs. 45is process is orchestrated by a complex network of pathways and factors that regulate gene expression and cellular behaviour. 46[50]

| CURRENT TRE ATMENT MODALITIE S
Advancements in technology have revolutionized cancer treatments and deepened our understanding of the biological processes involved.Various conventional and advanced methods are used to combat cancer. 51Treatment modalities have improved for better precision and effectiveness, leading to improved survival rates and enhanced quality of life of the patients.The treatment modalities include surgery, chemotherapy, radiation therapy, hormonal therapy, targeted therapy and more. 52Despite the significant progress in cancer treatment, chemotherapy continues to play a central role as a prominent alternative for advanced-stage malignancies where surgery or radiation may not be suitable due to specific circumstances. 53However, drug resistance remains a major challenge in cancer treatment, leading to increased instances of relapse and poor survival.

| Chemotherapy
Chemotherapy is a systemic approach widely employed for the treatment of malignant tumours, utilizing cytostatic drugs to target and halt the growth of cancer cells throughout the body for various cancers. 55This approach was inspired by the use of nitrogen mustard, a potent DNA alkylating agent. 56Currently, chemotherapy employs a diverse range of anticancer drugs with distinct mechanisms of action, inducing apoptosis by inhibiting cell division and precisely targeting cancer cells. 57Chemotherapy offers versatile options for cancer treatment, serving as a standalone therapy or synergizing with other oncological treatments in comprehensive approaches. 55The first is neoadjuvant chemotherapy, which is used to reduce the tumour size before surgery or radiation therapy and the second is adjuvant chemotherapy, which is used to destroy remnant cancer cells after surgery or radiation therapy. 58,59emotherapy drugs are classified as alkylating agents, antimetabolites, alkaloids, platinum derivatives and other natural products. 60The effect of chemotherapy is intricately linked to the cell cycle of cancer cells.The cytostatic drugs primarily target cells actively progressing through the cell cycle, disrupting the transition between different phases. 53

| Challenges in chemotherapy
While chemotherapy has demonstrated effectiveness in slowing tumour progression, it comes with a significant drawback: its impact on both cancerous and healthy cells leads to toxicity. 61Additionally, another substantial challenge is the emergence of resistance to chemotherapeutic drugs. 62Despite significant advancements in cancer treatment, overcoming resistance to chemotherapeutic drugs remains a major drawback.This adverse effect plays a pivotal role in disease relapse and contributes to poor survival rates in patients. 54emotherapeutic drug resistance can occur either by intrinsic or acquired mechanisms.Intrinsic mechanism is when cells acquire resistance against the drug at the onset of treatment.On the other hand, acquired mechanisms occur much later, during the course of cancer treatment. 63Emerging evidence indicates that microgravity can effectively modulate various cellular processes, including cytoskeletal alteration, reduced cell proliferation, altered gene expression and reduced invasion and metastasis. 64

| EFFEC T OF MI CROG R AVIT Y ON C AN CER PROG RE SS I ON
The cytoskeleton architecture plays an indispensable role in determining cell shape, facilitating cell migration and controlling cell survival and proliferation. 65The cytoskeletal protein consists of actin, intermediate filaments and microtubules which influence the cancer cell behaviour and contribute to the cellular processes critical for malignancy. 64,65[68][69] This intricate interplay between gravitational forces and the cytoskeleton plays a pivotal role in influencing essential cell functions such as proliferation, differentiation, remodelling of extracellular matrix and apoptosis. 70Cancer cells on exposure to microgravity form spheroids due to increased expression of actin, intermediate filaments and extracellular matrix components such as laminins, fibronectins, collagen and chondroitin sulphate. 71,72Studies have reported that reorganization and restructuring of actin filaments is very critical for cancer metastasis. 73 understand the impact of microgravity on structural and functional aspects of cells, Vassey et al. 74 investigated the effect of using mammary cancer cells (MCF-7), the results showed notable alteration in the nuclear proteins (Ki-67), a cell proliferation marker resulting in extended mitotic phase.Further, the cells also showed alteration in actin filaments and phosphotyrosine signal transduction.The MCF-7 cells also showed modification in the DNA distribution in the interphase cells, indicating the changes in the chromatin structure.All the effects have shown a profound influence on cell cycling, cytoskeletal dynamics, chromatin structure and gene expression. 74I G U R E 5 Schematic representation of (A) hindlimb unloading (B) tail suspension for rodent models. 289][80] Recently, Ahn et al. 81 investigated the effect of microgravity on the proliferation and migration of non-small-cell lung cancer (A549 and H1703).In both A549 and H1703 cells exposed to microgravity for 24 and 48 hours, the expression levels of MMP-2, MMP-9, TIMP-1 and TIMP-2 were enhanced.The findings indicate that microgravity promotes cell migration by modulating the ECM components.However, the migration and metastasis varies across different types of cancer. 81ng-term exposure to microgravity has a significant impact on signal transduction along the cytoskeleton to the nucleus and induces alteration in ECM proteins. 82Infanger and team 82 investigate the effect of the long-term influence of microgravity on papillary thyroid cancer cells (ONCO-DG-1) by exposing cells to the random positioning machine for 120 hours.The papillary thyroid cancer exhibited early alteration in the cytoskeleton and ECM proteins, triggering the formation of spheroids.Furthermore, enhanced accumulation of ECM components such as collagen type I and II, osteopontin, chondroitin sulfate, vimentin and fibronectin were observed.The levels of transforming growth factor beta-1 (TGF-β1) and its receptor (TGFBR2) were upregulated from 24 h until 120 h clinorotation, potentially contributing to ECM enhancement.TGF-β1 plays a significant role in cancer cell progression, exhibiting a dual role in both suppression and metastasis of cancer.In the early onset of the malignancy, TGF-β1 acts as a tumour suppressor and triggers apoptosis.However, in the advanced stage of malignancy, TGF-β1 promotes EMT invasion and metastasis. 83These results suggest that microgravity can potentially alter the TGF-β1 signalling pathway, leading to the suppression or metastasis of cancer. 84e impact of microgravity on poorly differentiated follicular thyroid cancer cells (TC) was determined by exposure to SMG in the CellBox-1 study and real microgravity in the International Space Station (ISS) during the CellBox-2 mission. 85,86The findings of the study revealed that TC cells showed distinct cell phenotypes with cells showing spheroid formation in response to microgravity.The results suggested that gravitational unloading in microgravity impacted various cancer cell processes, such as differentiation, proliferation, apoptosis, growth and invasion.Gene expression studies indicated that significant upregulation of ECM protein genes like COL1A1, potentially linked to cell detachment and spheroid formation, while downregulation of β1-integrin (ITGB1) suggested inhibition of cell adhesion.Altered gene expressions of cell adhesion factors (CAV1, ICAM1), growth factors (EGF, VEGFD) and inflammatory cytokines (IL-6, IL-8) were observed in space samples.Proto-oncogene Src (SRC) and focal adhesion molecule, vinculin (VCL) were downregulated, along with clear downregulation of key signalling pathways (NF-κB, ERK1/2). 87These interactions highlighted complex signalling relationships, indicating suppression of genes associated with growth, differentiation, proliferation, focal adhesion, progression and metastasis in spaceflight samples.Overall, the study suggests that F I G U R E 6 Schematic representation of microgravity simulation methods in humans. 29I G U R E 7 Stages of tumour development. 97icrogravity induces a redifferentiation of thyroid cancer cells and a shift towards a less-aggressive growth behaviour.
Cancer cell survival and metastasis majorly involve AKT activation. 88PTEN (Phosphatase and Tensin Homologue) suppresses AKT activation, preventing its nuclear translocation and further activating FOXO3 (Forkhead Box O3), resulting in tumour suppression. 88Recently, Arun and colleagues investigated the role of microgravity on PTEN/FOXO3/AKT pathway in colorectal cancer cells (DLD1, HCT116 and SW640). 89The results showed that all the CRC cells formed spheroids in response to microgravity, with distinct viability rates.Further, these cells showed distinct viability in response to microgravity.SW620 and HCT116 cells showed 20% viability, while DLD1 cells showed 40% viability under microgravity conditions.Gene expression studies indicated that cell death in response to microgravity was due to the upregulation of the tumour suppressors PTEN and FOXO3.This further led to the downregulation of AKT, triggering apoptosis by upregulation of CDKN2B and CDKN2D (CDK inhibitors). 89This study clearly indicates that microgravity regulates cell function by PTEN/FOXO3/ AKT pathway.
To understand the dysregulation in gene networks associated with the cell cycle, oncogenes and cancer progression triggered by microgravity, a genome-wide expression analysis was carried out in colorectal cancer cells (DLD-1) and lymphoblast leukaemic cells (MOULT-4). 90The findings revealed significant alterations in gene expression in response to microgravity.Specifically, DLD-1 cells exhibited upregulation of 1801 genes and downregulated 2542 genes, whereas MOULT-4 cells showed upregulation of 349 genes and downregulation of 444 genes.These changes induce alterations in the cytoskeleton, plasma membrane and intracellular signalling.Additionally, the study reported the dysregulation of the microRNA host genome, the microRNA-22 known for its tumour suppressor activity, displayed significant upregulation during microgravity exposure, potentially contributing to anti-proliferative effect. 90The migration and invasion of cancer cells is majorly regulated by Calcium ions (Ca 2+ ). 91Intracellular Ca 2+ plays a critical role in the reorganization of cytoskeletal proteins.Numerous calcium channels contribute to the invasion and migration of cancer cells, particularly store-operated calcium entry (SOCE), which has been reported to be closely linked to the invasion and migration of cancer cells. 92Recent study by Shi and team reported that microgravity significantly reduced the invasive and migratory capabilities of glioblastoma (U87 cells) by lowering the expression of calcium ion regulatory proteins such as ORA11, thereby inhibiting SOCE, resulting in a reduced influx of Ca 2+ into the cells. 93The findings of this study provide valuable insights into the relationship between microgravity and Ca 2+ ions and their impact on cellular processes.
The proteins mammalian target of rapamycin complex 1 (mTORC1), focal adhesion kinase (FAK) and ras homologue genefamily member A (RhoA) play a central role in maintaining cellular homeostasis.A recent study by Tan et al. 10 has reported that microgravity can significantly downregulate key signalling molecules, such as FAK, RhoA and mTORC1, in melanoma cells (BL6-10) which are involved in cell adhesion and migration. 10Further, the study also reported that microgravity activates Unc-51-like autophagy activating kinase 1 (ULK1) and AMP-activated protein kinase (AMPK) resulting in reduced proliferation and metastasis of melanoma.
Autophagy is a catabolic process that degrades cytoplasmic components in response to pathological stress.Ryu et al. 94 investigated the role of microgravity in regulating autophagy using GFP-LC3 cells.The results suggested that AMPK was activated in response to microgravity-induced cellular stress.These results suggest that prolonged exposure to microgravity can induce autophagy. 94

| EFFEC T OF MI CROG R AVIT Y ON CHEMOTHER APY
Microgravity has been reported to induce significant alterations in cellular shape, size, volume and adherence properties of cancer cells. 95However, understanding the molecular mechanisms is crucial for developing potential strategies to modulate cancer cells. 95To date, there are limited studies carried out to understand the behaviour of cancer cells in response to chemotherapy under microgravity conditions.Recently, Prashanth et al. 1 investigated the effect of microgravity on leukaemia cancer cells (HL40 and K562 cells) in response to chemotherapeutic agents (Doxorubicin and Daunorubicin).The findings demonstrate a notable influence of microgravity on cellular pathways, particularly ROS-sensitivity pathways and an increase in the effectiveness of drugs.However, the impact of microgravity on cellular pharmacological responses appears to be predominantly influenced by the specific cell type. 1 Cancer therapy involves the use of diverse drugs to target and eliminate cancer cells, however, the major challenge is the development of resistance to therapy due to the overexpression of multidrug resistance (MDR) proteins. 84A recent study by Rembiałkowska et al. 96 investigated the effect of microgravity in combination with chemotherapy (Doxorubicin) on the chemoresistant (EPG85-257 RDB) and sensitive (EPG85-257 P) gastric cancer cells.The results showed microgravity combined with chemotherapy showed decreased expression of drug resistance-related genes and increased marker in DNA/RNA damage.

| D ISCUSS I ON AND CON CLUS I ON S
Physical forces such as gravity and electromagnetic forces influence a wide range of biological processes and contribute to the overall function, development and maintenance of organisms.
Microgravity alteration encompasses variation in cell membranes, cytoskeletal rearrangement, limited proliferation and shift in protein and expression of genes, involved in the synthesis of gravitysensing proteins, proteins involved in cell differentiation, migration and signalling pathways associated with autophagy.These intricate interplays between microgravity and different biomolecules play a key role in regulating cell behaviour.
Microgravity can be simulated on Earth using various simulation methods and using these methods, we can potentially harness the effect of microgravity in targeting cancer.Chemotherapy, a widely used treatment strategy against cancer metastasis, faces a major challenge associated with the development of resistance.Hence, researchers are keen on developing alternative strategies in combination with chemotherapy to mitigate resistance to cancer.In our study, based on the previous research, we investigated the interplay between microgravity and chemotherapy on cancer cells.The altered gravity demonstrated a modulatory effect on the expression of various genes related to cytoskeleton proteins, metabolic pathways and ROS pathways.
In conclusion, our results suggest the application of microgravity in cancer treatment, showcasing its potential to increase cell sensitivity to chemotherapy.These observed alterations in cancer cells in response to microgravity could be harnessed for developing a promising avenue for the development of a new therapeutic strategy against cancer.Hence, researchers are keen on developing a strategy by utilizing chemotherapy in combination with SMG to combat the growth and proliferation of cancer cells.Earlier investigations have shown encouraging outcomes in both cell line and animal studies by enhancing the responsiveness of cancer cells to chemotherapy.Further comprehensive exploration of these findings in human subjects holds promise for refining and advancing this innovative approach in cancer treatment.

F I G U R E 3 1 F
Rotary cell culture system (RCCS) placed inside a CO 2 incubator.I G U R E 4 Schematic representation of diamagnetic levitation.